The invention relates to an encapsulating device, which is arranged to encapsulate a sample, in particular comprising at least one biological cell, in a polymer capsule, for example in an alginate capsule. Furthermore, the invention relates to a method for encapsulating a sample in a polymer capsule, in particular using the said encapsulating device. Application of the invention are available, in particular, in the encapsulation of biologically active samples, e.g. biological cells such as, for example, pluripotent stem cells or primary-tissue cells, in biocompatible polymers such as, for example, alginate.
It is generally known to immobilize biologically active substances such as, for example, enzymes, proteins, biological cells, cell components or cell groups, for applications in chemical, biochemical or medical procedures. The immobilization may include completely covering (encapsulating) the substance in a matrix material (encapsulating substance). For example, for applications in regenerative medicine, it is known to embed cells, tissue or micro-organs, as transplants, in a biocompatible encapsulating substance. The encapsulation offers the possibility, advantageously, of protecting the transplants against an immune response of a recipient, and against mechanical stress, while, at the same time, ensuring that the supply of nutrient to the transplant is maintained.
Frequently, encapsulation in three-dimensional, for example spherical, capsules is of interest, since, for biological cells, the three-dimensional environment is similar to a physiological environment, so that the cells keep better than in a layer-type encapsulation, for example on the surface of a substrate. For example, it is known that only in a three-dimensional environment do chondrocytes produce the collagen typical to the formation of cartilage, this being one reason for the delayed healing of cartilage defects.
Frequently used as an encapsulating substance is the polymer alginate. Alginate, besides cellulose, is a principal structural component of the cell walls of marine brown algae (phaeophyceae). Alginate is an acid polysaccharide, composed of 1-4-linked α-L-guluronic acid and β-D-mannuronic acid chains. Homopolymer regions (MM blocks or GG blocks) and heteropolymer regions (MG blocks) are formed. The carboxyl groups of the acid chains can be cross-linked by bivalent cations (for example, Ca2+, Ba2+, Fe2+). In the cross-linked state, alginate forms a hydrogel, which has proved to be advantageous for encapsulating biologically active substances. The viscosity of the cross-linked alginate increases as the concentration of cations increases, and alginate cross-linked with calcium or barium is stable at physiological temperatures.
Various methods are known for encapsulating, for example, biological cells in alginate. For example, it is known to generate drops of a suspension of biological cells in an alginate solution by means of compressed air, and to move them into a bath of a polymerization substance having bivalent cations, where cross-linking of the alginate chains occurs (see U. Zimmermann et al. in “Ann. N.Y. Acad. Sci.”, volume 944, 2001, pp. 199-215). In the case of a further known method, falling drops of a cell-alginate suspension are bombarded with crystallites containing bivalent cations, thereby promoting internal cross-linking of the drops (see H. Zimmermann et al. in “Biomaterials” volume 28, 2007, pp. 1327-1345).
The conventional methods for encapsulating cells have several disadvantages. Firstly, the conventional techniques are distinguished by a high consumption of cells and alginate solution. In the generation of the falling suspension drops, the desired cell, or the desired number of cells, is not contained in each drop. The reproducibility of the encapsulation is dependent on numerous procedure conditions, and particularly on the capabilities of the person performing the encapsulation. Finally, there are limitations in combining the conventional encapsulation methods with complex processes in the cultivation (propagation, growth and/or differentiation) of biological cells. The latter is of importance, in particular, in the handling of therapeutically relevant cells such as, for example, stem cells. For the encapsulation of stem cells, in particular, there is an interest in three-dimensional embedding, since this three-dimensional embedding can be expected to have advantages for the proliferation and development of the cells.
A further known application of encapsulation in biocompatible polymers, for example alginate, is that of cryoconservation. The protective covering of the cells can reduce mechanical stress caused by any extra-cellular ice crystal formation and, in the case of cryoconservation of cell aggregates, cell-to-cell contacts can be protected.
Conventional immobilization techniques are not limited to the encapsulation of biologically active substances in alginate. Likewise, in other tasks of encapsulating substances in polymers, such as, for example, insulin in chitosan, there is an interest in embedding in three-dimensional, for example spherical or drop-shaped, capsules.
Furthermore, it is known to cultivate cells in hanging droplets, e.g. for the defined formation and cultivation of three-dimensional, multicellular aggregates. Hitherto, the conventional techniques have been limited exclusively to the cultivation of the cells in the hanging droplets.
It is the objective of the invention to provide an improved encapsulation device, with which disadvantages of conventional techniques are avoided. The encapsulation device is intended, in particular, to enable encapsulation with improved yield, increased reproducibility and/or reduced stress for the encapsulated sample. Furthermore, specifically, there is interest in an improved encapsulation device that is suitable for encapsulating samples comprising at least one biological cell, and that simplifies integration into processes for handling biological cells. It is a further objective of the invention to provide an improved method for encapsulating a sample in a polymer capsule, with which disadvantages of conventional methods are avoided. The encapsulation method is to be distinguished by an increased yield, improved reproducibility and/or reduced stress on the encapsulated samples.
These objectives are achieved by an encapsulation device and/or an encapsulation method having the features of the invention.